Biosensor surface structures and methods

Abstract

A biosensor surface with a low density of ligand-carrying tether molecules on a base layer. Also, surface plasmon resonance (SPR) devices have the biosensor surface attached to a thin gold layer with backside angle-spread incident radiation for resonance excitation and reflective detection.

1. A process of fabricating a sensing surface, comprising the steps of:
 (a) forming a base layer, said base layer is composed of a monolayer of colloidal particles chosen from a group consisting of proteins, peptides, colloidal PTFE particles, colloidal polyethylene particles, colloidal polypropylene particles wherein said particles are of a diameter in the range of 0.5 nm to 100 nm; 
 (b) epoxy-group activating said monolayer by covalent coupling of a monolayer of a bisoxirane, said epoxy-group activated monolayer is of a thickness not to exceed 1 nm; 
 (c) reacting a fraction of said epoxy groups with thioglycolate to incorporate carboxyl groups and the remaining fraction are hydrolyzed to yield hydroxyl groups; and 
 (d) binding ligands to said monolayer. 
 
     
     
       2. The method of  claim 1  wherein said monolayer of colloidal particles is activated by exposure to an oxygen gas plasma for the covalent incorporation of hydroxyl groups and peroxide groups wherein said monolayer of bisoxirane is covalently bound via said reactive oxygen containing groups. 
     
     
       3. The method of  claim 1  wherein said colloidal particles are proteins or peptides and wherein functional groups contained on surface of said protein or peptide molecules, are chemically derivatized to yield reactive thiol groups. 
     
     
       4. The method of  claim 3  where said derivatization of said functional groups is accomplished using the thiolating reagents 2-iminothiolane or N-acetylhomocysteine thiolactone. 
     
     
       5. The method of  claim 1  where said bisoxirane is butanedioldiglycidyl ether. 
     
     
       6. The method of  claim 1  wherein carboxylic groups and hydroxyl groups are incorporated onto said epoxy activated monolayer by reaction with sodium thioglycolate under alkaline aqueous conditions. 
     
     
       7. The method of  claim 1 , wherein said ligands possess one or more tether molecules of a length exceeding 1 nm wherein said tether molecules contain at least one reactive group that forms an irreversible covalent bond with said base layer reactive groups. 
     
     
       8. An SPR biosensor, comprising:
 (a) a base layer bound to a plasmon resonance capable metal film, said base layer composed of a monolayer of colloidal particles chosen from a group consisting of proteins, peptides, colloidal PTFE particles, colloidal polyethylene particles, and colloidal polypropylene particles, said particles are of a diameter in the range of 0.5 nm to 100 nm; 
 (b) ligands bound to said base layer; and 
 (c) an optical source and reflection detector optically coupled to said metal film; 
 (d) wherein said monolayer of particles is epoxy group activated by covalent coupling of a monolayer of a bisoxirane, said epoxy-activated monolayer is of a thickness not to exceed 1 nm.